The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics and post-translational modifications. The following major findings were made over the last year: 1) We have previously shown that protein phosphorylation in the mitochondria matrix is extensive. Using minimally disruptive blue native gel electrophoresis we have demonstrated that much of the kinase activity phosphorylating mitochondria oxidative phosphorylation complexes was identified to exist within the Complex itself. That is, when the different complexes were purified by blue native electrophoresis, the protein kinase activity within the complex was maintained. This implies that mitochondrial kinases are compartmentalized in the protein assemblies making up the oxidative phosphorylation complexes. Antibody screening suggested that typical cytosolic protein kinases (PKA, PKC etc) are not present in these complexes, suggesting that the complexes themselves have unique protein kinase activity. 2) Quantitative matrix protein phosphorylation screens using iTraq labeling and mass spectroscopy for protein and protein phosphorylation identification have identified the sites and quantitiative changes in phosphorylation state during different physiological conditions, such as calcium levels or energy state of the mitochondrion. Many of these sites are novel discoveries that are now being exploited to examine their role in energy metabolism regulation. 4) A hypothesis has been developed that tissues with large swings in metabolic activity (i.e. heart and muscle) hold mitochondrial electron transport activity in reserve via post translational modifications while more constant activity tissues (i.e. liver) have most of their enzymatic activity active and available. We also propose that this relationship holds in animals of different sizes where the heart has very high (man, large animals) or low (mouse, rat, small animals) dynamic range due to their relative differences in basal activity. We have demonstrated that the activity of mitochondria Complex V and IV indeed follow this notion, being suppressed in heart and skeletal muscle of pigs while fully active in the porcine liver. In contrast, the constantly active mouse heart and liver have essentially identical enzyme activities. An intermediate animal, the rabbit, showed intermediate values between the pig and mouse. These studies suggest that an inherent post-translational throttle on oxidative phosphorylation enzymes is present likely preventing the buildup of potential energy and associated reactive oxygen species under resting conditions. 5) Phosphate binding to Succinyl-Coa Synthetase (SCS) enzyme of Krebs cycle was shown to activate SCS in a classical V-type allosteric mechanism enhancing the Vmax of this enzyme. Phosphate signaling through this SCS may influence the ability of phosphate to stimulate the generation of NADH from Krebs Cycle but also the substrate level generation of ATP during oxygen deprivation.